Characterization of post-translational modifications (PTM) complement is critical to understand the normal protein function across biology and its disruption by diseases. To help map the PTM pattern that defines the epigenetic code, we propose to separate the isomers with different PTM localizations for human histones and histone tails using high resolution FAIMS, linear IMS, and eventually their 2-D combination. This approach will extend to all proteins.

Our goal is to develop a capability to separate isomers of intact proteins and their large pieces with PTMs in different positions that often have distinct in vivo activities. While such localization variants involving various PTMs are ubiquitous in life, they are especially prevalent and important for histones where they constitute a combinatorial epigenetic code. Hence the project focuses on core histones, but the findings would transfer to all proteins.

These variants have been analyzed using bottom-up proteomics, after proteolytic digestion and chromatographic separation of resulting isomeric peptides. Those methods fail to map the crucial complete PTM pattern because of incomplete sequence coverage and limited variant separation. Variants can also be identified for whole proteins (top-down) or their large pieces (middle-down) employing electron transfer dissociation (ETD), but the lack of means to separate them prior to mass spectrometry (MS) has obstructed analyses of commonly encountered isomeric mixtures. We aim to establish an approach to separate the variants on the protein and large peptide level using high-definition differential ion mobility spectrometry (FAIMS) before the MS stage. This effort will utilize FAIMS with recently introduced buffers rich in helium or hydrogen, which has initially resolved the localization variants of full human histone tails up to 5 - 6 kDa.

Our demonstrated baseline separation of 3-D conformers for ~10 - 15 kDa proteins suggests the feasibility of resolving the variants for similarly-sized histones. Here, we will optimize the separations of variants for full tails (methylated, acetylated, or phosphorylated on specific sites) and expand it to cellular extracts. The separated variants will be identified by MS/MS using ETD or synthetic standards, enabling subsequent assignment based on the catalogued peak positions. The same samples will be probed using a traveling-wave IMS/ToF platform to evaluate the resolution in linear IMS dimension and assess the conformational differences underlying the IMS and FAIMS separations. This information will allow projecting the utility of 2-D FAIMS/IMS analyses for histone proteoforms contemplated as a follow-up to this pilot project.